Deciphering the role of low complexity domains in dual specificity kinase function

项目摘要 双特异性蛋白激酶在真核生物中发挥着不可或缺的作用，包括 信号转导通路的调节和亚细胞区划。从结构上讲，在 除了它们折叠的激酶结构域，它们还由相当长的片段组成，这些片段是 本质上是混乱的。鉴于这些激酶的折叠结构域被广泛研究 (从生化和结构上)，人们对无序分子的功能作用知之甚少 显示出偏向极性和带电氨基酸的组成偏向的结构域。 在我们对所有人类双特异性激酶的初步序列分析中，我们发现 CLK3具有最长的本征无序区。最近的研究表明CLK3是连锁的 多种癌症病理和乳糜泻。例如，CLK3的过度表达是 与骨肉瘤、肝癌和胆道癌有关。CLK3基因的序列变异 与膀胱癌、克罗恩病和多种乳糜泻有关。在功能上， CLK3被募集到核斑点中，在调控Pre-mRNA剪接中发挥重要作用。 在这项拨款申请中，我们建议表征序列决定因素，分子 体外联合使用CLK3核斑点招募的密码和功能 以及活体实验。根据我们的初步分析，我们假设(I)低复杂性 CLK3的无序结构域(残基1-285)驱动蛋白质的核斑点定位，以及 (Ii)CLK3斑点定位/动力学的改变与疾病病理有关。我们 将利用综合的生物物理、生化和细胞生物学来检验这些假说 接近。为此，我们将利用剪接变异体，疾病连锁突变体，并理性地 扰乱了LCD变体，系统地破译了LCD在CLK3的S功能/功能障碍中的作用。 成功完成拟议的研究不仅将阐明这种无序的作用 CLK3(病态)生物学中的结构域，但也提供了对 LCD和KK结构域中的双特异性激酶功能和功能障碍。

Project Summary Dual specificity protein kinases perform indispensable roles in eukaryotes including the regulation of signal transduction pathways and subcellular compartmentalization. Structurally, in addition to their folded kinase domains, they are composed of significantly long segments that are intrinsically disordered. Whereas the folded domains of these kinases are studied extensively (both biochemically and structurally), little is known about the functional role of the disordered domains that display a compositional bias towards polar and charged amino acids. In our preliminary sequence analysis of all human dual specificity kinases, we identify that CLK3 has the longest intrinsically disordered region. Recent advances indicate that CLK3 is linked to multiple cancer pathologies and celiac disorders. For example, overexpression of CLK3 is associated with osteosarcoma, liver cancer and bile cancer. Sequence variations in CLK3 gene is associated with bladder cancer, Crohn’s disease and multiple celiac disorders. Functionally, CLK3 is recruited to nuclear speckles and plays vital roles in the regulation of pre-mRNA splicing. In this grant application, we propose to characterize the sequence determinants, molecular codes and functional roles of CLK3 nuclear speckle recruitment utilizing a combination of in vitro and in vivo experiments. Based on our preliminary analyses, we posit that (i) the low-complexity disordered domain of CLK3 (residue 1-285) drives the protein’s nuclear speckle localization, and (ii) alterations in CLK3 speckle localization/dynamics is associated with disease pathologies. We will test these hypotheses utilizing an integrative biophysical, biochemical and cell biology approach. To this end, we will utilize a splice variant, a disease-linked mutant, and rationally perturbed LCD variants to systematically decipher the LCD’s role in CLK3’s function/dysfunction. Successful completion of the proposed study will not only illuminate the role of this disordered domain in CLK3 (patho)biology, but also provide significant insights into the interplay between the LCDs and kinase domains in dual specificity kinase functions and dysfunctions.